Effect of Methyl Methanesulfonate on Macromolecular Biosynthesis in P388F Cells

[CANCER RESEARCH 27 Part 1, 2134-2239, November 1967} Effect of Methyl Methanesulfonate on Macromolecular Biosynthesis in P388F Cells

BRIAN W. FOX AND MARGARET FOX Paterson Laboratories, Christie Hospital, Withinglon, Manchester 20, England

SUMMARY (7, 8), and animal tumors (8, 14), there must be many potential sites for primar}' action. In the case of nitrogen mustard, apart The effect of methyl methanesulfonate, ethyl methanesulfonate, from effects on DNA itself, a nonhistone protein has been im isopropyl methanesulfonate, and mÃ©thylÃ¨nedimethanesulfonate plicated in a reaction concerned with inhibition of the primer on the survival of the mouse lymphoma line, P388F, is described. activity of DNA alkylated in vivo (28). The concentrations of alkanesulfonate resulting in a 507c inhibi The aim of this communication is to present some data tion of growth after a 3-hr exposure were 0.1 HIM,>16 mM, >1.5 obtained using the second, labeled precursor approach in relation imi, and 0.037 m.M,respectively. to the elucidation of the priman- biologic action of methyl A more detailed examination of the action of methyl methane methanesulfonate and related alkanesulfonates in the P388 sulfonate with regard to the time of onset of inhibition of nucleic lymphoma system. acid and protein synthesis was made. The relative uptakes of thymidine-3H, uridine-5-3H, glycine-3H, and tyrosine-3H were investigated and compared. Methyl methanesulfonate affects dif MATERIALS AND METHODS ferentially the ability of the cell membrane to transport different Cell Line. The cells used in these experiments were primary macromolecule precursors into the cell pool. The potential causes expiants of the murine lymphoma line, P388F, derived from the of this differentia] action are discussed in relation to the known P388 line, previously described (10). The cells were harvested sensitivity to alkylation of the guanine bases in DNA and from the peritoneal cavity of DBA2 mice, about 14 days after a RNA. A mechanism involving the codon-anticodon structure of parenteral injection of IO6cells. The cells were diluted, immedi the amino acyl transfer RXA and its recognition is proposed. ately after removal from the peritoneum of the mouse, in Fischer's medium supplemented with 10% horse serum, to a con INTRODUCTION centration of 2.5 X IO6cells per ml for the uptake experiments. The suspension was allowed to equilibrate at 37Â°Cin a water Two general approaches to the study of the biochemical changes induced by alkylating agents have been employed: (a) bath for 2 hr before the commencement of the experiment. the labeled drug method, in which the transferred alkyl group Chemicals Used. Alkyl alkanesulfonates were synthesized is isotopically labeled and the treated biologic tissue analyzed in by conventional procedures and purified as described pre detail for labeled products; and (Ã¨)the labeled precursor method, viously (15). All solutions were made up immediately before use and kept ice-cold to avoid hydrolysis. Thymidine-3H and labeled which employs an unlabeled alkylating agent and labeled nucleic acid and protein precursors which are administered to the bio amino acids were obtained from the Radiochemieal Centre, logic system before, during, of after drug treatment. The resulting Amersham, England. tissue is then examined for the disposition of the precursor and Dose Response Studies. The general procedure adopted in its extent of incorporation with reference to control experiments. the uptake studies in relation to dose of alkyl alkanesulfonate was as follows. Sterile, glass-stoppered tubes (total capacity, 15 The labeled drug approach has been extensively used with certain alkylating agents, notably sulfur and nitrogen mustards ml) with a small protrusion blown outwards about 1 cm below (29) ethyleneimine (21, 27), diazoalkanes (20), and alkanesul- the base of the stopper, were used. Two ml of cell suspension (2.5 X 106/ml) were added to each tube. Stock solutions of fonates (17). Apart from detoxification data (24, 25), this approach has revealed that nucleic acids are preferentially alkyl- alkanesulfonates in growth medium were adjusted so that the ated in X-7 position of guanine and that there is a lesser degree required dose could be given to the cultures by addition of 0.1- of alkylation of N-l cytosine, N-l adenine, and N-3 adenine ml volumes. A solution of labeled precursor in 0.05 ml was added (17). Based on such studies, the primary action of the bifunctional to each side bulb and the tubes stoppered. After exactly 30 min mustards has been considered to be the cross-linking of DNA of incubation without shaking, the tubes were inverted, mixing between two guanine moieties (18). It is clearly not possible to the isotope with the cell cultures. Two hr or an appropriate time offer this interpretation for monof'unctional alkylating agents, later, the tubes were plunged in an ice bath and ice-cold buffered and, in view of the wide diversity of biologic action which the saline, pH 7.0 (2 ml), added. The cells were subsequently cen- alkanesulfonates exhibit in spermatogenesis (8, 15), hemopoiesis trifuged (2000 X g, 7 min) and washed twice with buffered saline. Time Course Studies. Suspensions of cells (2.6 X 10"cells Received March 31, 190ÃŒ7;acceptedJuly 14, 1907. per ml) (25 ml) were incubated at 37Â°Cwith appropriate con-

2234 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1967 American Association for Cancer Research. Methyl Methanesulfonale centrations of the alkanesulfonates (1 ml). At different times able for the two further extractions with hot TCA. The washings after the addition of the alkylating agent, aliquots (2 ml) were from each tube were again combined (hot TCA extracts). The pipeted rapidly into 15-ml glass-stoppered tubes containing residue was washed twice with water, 2 washings of 2 ml each isotopically labeled precursor in aqueous solution (0.05 ml). (water extract), and the final residue drained by inverting the After 15-min incubation, the tubes were removed and cooled in tubes over filter paper for 5 min. ice. Ice-cooled buffered saline was then added as before. The Residues were treated with Pronase R (0.2 ml of a 1 mg/ml cells were washed and centrifuged twice more before processing. solution in sodium phosphate buffer; 0.05 M, pH 6.5) and incu bated overnight. The entire contents of the tube were then dis Isolation of High-Polymer DNA solved in a scintillation mixture (10 ml) consisting of naphtha lene (100 gm), toluene (700 ml), 2,5-diphenyloxazole (1 gm), Detergent Method. Pellets of washed cells were treated with l,4[2'(5'phenyloxazolyl)]benzene (25 mg), and dioxane (700 ml). citrate-buffered saline (0.8 ml) and homogenized in the cone- The TCA extracts were pipeted (0.2 ml) on to glass fiber discs shaped centrifuge tubes with a Perspex pestle made to fit closely (Whatman GF/A, 2.1 cm) which were then dried for 25 min to the conic end of a centrifuge tube (10 ml). A solution of so under an infrared lam)). For counting, several discs were com dium dodecyl sulfate (0.2 ml, 5% in 45% ethyl alcohol) was bined together in the same scintillation solution, By separate added and the mixture allowed to stand at room temperature experiment, it can be shown that the total counts measured on for 1 hr with occasional shaking. A solution of 5 M NaCl (0.21 a series of discs in the same phosphor is stoichiometrically re ml) was added to each, followed by 1.2 ml of 95% ethyl alcohol. lated to the number of discs present (J. W. Davies, personal The DNA was spooled off on thin glass rods and dissolved in communication). Where activity was low, TCA was removed sodium acetate solution (1.5 ml, 0.2%). It was allowed to stand by extraction with ether, then the solutions were lyophilized and for approximately 2 hr to allow solution, then sodium acetate transferred to glass fiber discs described above. There was no (0.16 ml, 40%) was added, and the resulting mixture extracted advantage in washing the discs with ether before counting to with Â«-butylalcohol :chloroform (1.6ml, 1:3). This mixture was remove TCA. Reliable and reproducible results were obtained then centrifuged (2000 X g for 30 min at 5Â°C)and the top clear with the above procedure with the number of cells used. layer removed. Ethyl alcohol (95%, 2.0 ml) was then added and The instruments used were the Beckman liquid scintillation the DNA spooled off and again suspended in sodium acetate counter and both the two-channel external standardization solution (1.0 ml, 0.2%). In some cases, UNA was removed by method on this instrument and an internal standard of toluene- incubating with ribonuclease (heat-treated, 100Â°Cfor 10 min) 3H (25 fie/ml, 0.01 ml) were used to determine the amount of and reprecipitated. quenching. In counting treated residues, quenching rarely ex Phenol Method. A solution of sodium dodecyl sulfate (1%) ceeded 20%. and sodium paraminosalicylate (5%) in 4 ml water and phenol: wi-cresol:9-hydroxyquinoline:H2O (500 gm: 75 ml: 0.5 gm: RESULTS 55 ml) (4 ml) was mixed with the washed cell pellet, and the whole mixture shaken and allowed to stand overnight. After Effect of Alkanesulfonates on Cell Survival centrifugation (10,000 X g for 30 min at 5Â°C),the upper layer was removed and sodium chloride solution (30%, 0.1 ml) was The relative effects of 4 alkanesulfonates on the survival of then added to make the final concentration 3% with regard to P388 cells were measured by technics described previously (9, 11) and the results are shown in Chart 1. MÃ©thylÃ¨nedimethane- salt. The solution was then thoroughly mixed, and phenol (88%, 0.5 volume) added; after further mixing and centrifugation sulfonate was the most effective of the four drugs and the doses (13,000 X g, 10 min), the top phase was removed and treated required to reduce survivors to 50% of control in each case were mÃ©thylÃ¨nedimethanesulforiate (0.037 min), methyl methane- with ethoxyethanol (1 volume). The DNA formed was spooled off, collected in sodium acetate, and reprecipitated by the method sulfonate (0.1 HIM),isopropyl methanesulfonate (>1.5 HIM),and ethyl methanesulfonate (>16 HIM).The relative hydrolysis rates described above. of these esters at 37CC in aqueous media were 22 min, 9.1 hr, Analytic Methods. The diphenylamine procedure of Burton (2) was used to estimate the DNA concentration; RNA and 13.6 min, and 10.4 hr, respectively (26). protein contamination was estimated by the methods of Ceri- otti (3) and Lowry et al. (19), respectively. Time Course of Inhibition of Thymidine-3H Uptake into Trichloracetic Acid Separations. Washed cell pellets were DNA after Treatment of P388 Cells with Methyl Methane treated with ice-cold trichloracetic acid (TCA) (5%, 2.0 ml) and sulfonate sonicated for 25 sec in an MSE ultrasonic generator. The sample was maintained in an ice bath so that the temperature did not As can be seen from Chart 2, the time of 50% inhibition of rise above 5Â°C.Subsequently, tubes were centrifuged (2,000 X thymidine-3H uptake into DNA during exposure to a concen g, 5 min), the residue similarly extracted 3 times, and the wash tration of 0.91 HIMmethyl methanesulfonate is about 0.75 hr. ings from each collected together (cold TCA extracts). A solu tion of TCA (5%, 2.0 ml) was added to the residue from the Time Course of Inhibition of Protein and Ribonucleic last cold TCA extraction and the tube incubated in a boiling Acid Synthesis by Methyl Methanesulfonate water bath for 8 min, with a glass marble on the opening to re duce evaporation. The mixture was then cooled and again ultra- The relative uptakes of labeled amino acids into the protein sonicated (1 min) to break the precipitate into a fine form suit residues of TCA-extracted cells at different times after treat-

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1967 American Association for Cancer Research. Brian W. Fox and Margaret Fox ment with methyl methanesulfonate are shown in Chart 3. The uptake of uridine-5-3H is also shown for comparison. It can be seen that 50% inhibition of RNA synthesis occurs about 100-0- 1.1 hr after addition of the drug (0.91 HIM). The relative up takes of different amino acids at this dose level are shown in Table 1 together with the environmental amino acid concen tration. The influence of the concentration of the amino acid in the surrounding medium was studied in the case of glycine by O comparing the uptake of a tracer level of glycine in medium with te. and without glycine supplementation. In unsupplemented me dium, the glycine concentration was 12.01 /ig/cell suspension o u (2.15 ml) and in the supplemented medium, 48.85 Mg/cell sus 'S 50- pension. The fourfold difference in concentration failed to affect either the uptake of labeled amino acid into the free form within UJ

I 0-

â€¢O- :O â€” i i O 0.5 1.0 1.5 HOURS AFTER ADDITION OF MMS CHART 2. Effect of methyl methanesulfonate (MMS) (0.91 HIM) on the uptake of thymidine-'H (15-min exposure) into deoxyribonucleic acid at different times after addition of alkylating agent (cell concentration 2.5 X IO6cells/ml).

Effect of Methyl Methanesulfonate on the Kates of In corporation of Labeled Amino Acids into the Free and RNA- bound Forms within the Cell The free amino acid within the cell was taken to be related directly to the ice-cold 5% trichloracetic acid fraction of the cells, while the hot TCA extract would give some indication of the extent of binding of the amino acid to the hot TCA-extrac- table nucleic acids, i.e., RXA (presumably the aminoacyl transfer RNA) and DNA containing traces of amino acids. The relative uptakes of glycine-3H, tyrosine-'H, and uridine-5-3H were inves tigated after methyl methanesulfonate. The tyrosine uptake into the cell pool increased to 135% of the control, while at the same drug concentration uridine-5-3H uptake into the nucleotide pool was reduced to 25% of the control values (Chart 4). The hot trichloracetic acid extract radioactivity from the tyrosine experiment is increased at the lower concentrations but returns to normal levels at the higher doses of methyl methane sulfonate (Chart 5). The effect of methyl methanesulfonate on the glycine levels is less marked. The uridine level in this fraction T isdecreased and isinversely related to the dose of methyl methane 0.2 0.4 06 08 sulfonate re]Â¡resentingan inhibitory effect on RNA synthesis. DRUG CONCENTRATION (mM) The residue radioactivity (Chart 6), representing protein synthesis, shows that uptake of tyrosine is less affected than CHART 1. Relationship of surviving fraction of P388F lymphoma that of glycine. Since the percentage uptake into protein is in cells exposed for 3 hr to different concentrations of mÃ©thylÃ¨ne dependent of the specific activity of the amino acid in the me dimethanesulfonate (MDMS), methyl methanesulfonate (MMS), dium, this difference may represent a difference in sensitivity of ethyl methanesnlfonate (EMS), and isopropyl methanesulfo the protein-synthesizing system to methyl methanesulfonate for nate (IMS) in Fischer's medium (with 10% horse serum). the two amino acid species.

2236 CANCER RESEARCH VOL. 27

Txrosine

100-** Glycine

O ce.

O U Glycine

^""Aâ€” Alanine 111 u \ â€¢â€¢-â€¢Tyrosme te 111 ^^_ RNA synthesis (L p .0 1.5 2.0 2.5 HOURS AFTER ADDITION OF DRUG CHART 3. Effect of methyl methanesulfonate (0.91 HIM) on the Uridine uptake of glycine-3H, alaniue-'H, tyrosine-'H into protein, and uridine-5-3H into ribonucleic acids, at different times after addi tion of alkylating agents (cell concentration and precursor expo sure times as in Chart 2). 1.2 1.6 DRUG CONCENTRATION (mM) TABLE 1 CHART 4. Effect of methyl methanesnlfonate at different con Effect of Methyl Methanesulfonate (0.01 m\i) on the centrations on the uptake of tyrosine-3H, glycine-3H, and uiidine- Rate of Inhibition of Precursor Incorporation 5-3H into the cold trichloracetic-acid-solvible pool of P388F cells. into Macromolecule Exposure times: alkylating agent 0.5 hr followed by precursor inhibition of for 2 hr in the continued presence of alkylating agent (cell con PrecursorThymidine-'H time(min)45 precursor(mM)0.023 centration 2.5 X IO6 cells/ml). X IO"6 TJridine-3HGlycine-3H 6810179 0.023 X10-Â«0.017 acid precursor under normal circumstances. Under these condi tions, a reduction in precursor uptake would be due to 1 of 4 Arginine-3H 0.074 factors: an interference with deoxyribonueleic acid synthesis, a Proline-3HAlanine-3HTyrosine-3H50%726947Concentration0.02980.0290.305 reduction in cells synthesizing DXA, a breakdown of labeled DNA after treatment, or enzymatic failure to utilize preformed precursor present as nucleoside. There is evidence that at the higher drug concentrations in these studies all these systems may contribute to the final result. The drug is also effective in pro DISCUSSION ducing marked changes in the cell kinetics at lower treatment Alkanesulfonates, both mono- and bifunctional, have been levels (9), and it is clear that other subtle and important changes shown to induce a variety of biologic effects (1) in whole animals occur at such levels which may not be directly related to the (26, 27) and in cell culture systems (4,5). action of the drug on DNA but to some other controlling factor From comparative work with a number of simple alkyl alkane- in the general cell metabolism. sulfonates in spermatogenesis (15), it became clear that the To examine the nature of reactions occurring after treatment alkylating alkyl group is the main feature which differentiates with methyl methanesulfonate, the levels of free precursor, one agent from another, and that the composition of the alkane precursor initially bound within the cell, and radioactivity of sulfonic acid used in its synthesis played little or no part in de the final macromolecule under consideration were investigated termining the character of its biologic activity. using both the phenol extraction procedure and a modified A comparison of the lethality of 4 alkanesulfonates in P388F Schmidt-Thannhauser method. \Yhen an amino acid precursor cells is shown in Chart 1 and is not related to the hydrolytic half- was used in the latter procedure, the "5% cold trichloracetic life of the esters in aqueous solution. The exposure time was acid" extract was taken to be a measure of the quantity of free chosen to allow about 75% of the cells to take up labeled nucleic amino acid present in the cell pool. The "hot TCA extract" was

NOVEMBER 1967 2237

Tyrosine

O DC K O U 'S 50-

0.4 0.8 1.7 1.6 DRUG CONCENTRATION (mM) CHART 6. Effect of methyl methnnesulfonate at different con centrations on the residue (protein) uptake of tyrosine-3H and glycine-3!!. Conditions of exposure times and cell concentrations as in Chart 4. 0.4 0.8 1.2 1.6 study, however, no attempt has been made at this stage to iden DRUG CONCENTRATION (mM) tify which of these routes are likely to be affected. CHART 5. Effect of methyl methaiiesiilfonate at different con By comparison of the tyrosine-3H, glycine-'H, and uridine-5- centrations on the uptake of tyrosine-'H, glycine-3H, and uridine- 3H uptakes into the various fractions of the cell at different dose 5-3H into the hot trichloracetic acid extract of P38SF cells follow levels of methyl methanesulfonate, it is clear that these pre ing removal of the cold acid-soluble pool. Exposure times and cell cursors are differentially affected in their entry into the cell pool concentration as in Chart 4. (Chart 4). Whereas the free ty rosine-3H within the cell is in creased at the higher dose levels of methyl methanesulfonate, considered to isolate the aminoacyl transfer RNA radioactivity uridine-5-3H by contrast is decreased. This study suggests that together with any small oligopeptides that may be soluble in this there is not an indiscriminate increase in permeability to all pre TCA fraction. The "residue" was regarded as protein and poly- cursors, but a marked differential sensitivity. peptide. Clearly, many factors operate in determining the nature of In Streptococcusfaecalis (22), protein synthesis was found to be this differential effect. The simplest interpretation would be a a function of the concentration of membrane-associated poly- direct effect on one or more of the transport systems across the ribosomes, and in Bacillus slearothermophilus, the cell membrane cell membrane. However, an alteration of the rate of utilization was shown to be the most important initial site of amino acid of the pool precursor would also bring about differences in pool incorporation in the form of an aminoacyl transfer RXA (1). size as well as a disturbance of the alternative pathways of de The highest activity after exposure to a labeled amino acid oc novo and preformed precursor utilization. In the particular case curred in the lipoprotein fraction, 30 seconds after addition of the of amino acid uptake, inhibition of the formation of aminoacyl amino acid. This was followed very rapidly by a labeling of the transfer RNA as well as an inhibition of one of the several events membrane-associated aminoacyl transfer RXA. involved in the incorporation from aminoacyl transfer RNA to It is implied, therefore, that the mechanism of uptake of the developing protein on the polyribosome could lead to such amino acids into a cell is very carefully controlled at the cell mem an increase. Supplementation of the medium with glycine does brane and that the initial mechanism for protein synthesis occur not alter the rate of uptake inhibition or its equilibration within at this site, at least in bacterial cells. There is now an increasing the cell. volume of data accumulating (4,5) concerning the transport In view of the fact that the guanine base is known to be 2 to mechanisms involved across the cell membrane in mammalian 3 times more sensitive to the action of certain alkylating agents (23, 26), yeast (12, 13), and bacterial (1, 22) cells. Three main than other bases, such as adenine and cytosine, in the nucleic routes have been recognized (16), and several of the factors in acid (17), it seems reasonable to conclude that the codon-anti- volved in each route have been elucidated (6, 16). In the present codon combination which determines the type of amino acid

2238 CAXCEH 1ÃŽESEAUCH VOL. 27

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1967 American Association for Cancer Research. Methyl Methanesulfonatc transferred to the protein-synthesizing site may have a sensi cerevisiae. I. Evidence for a Specific Arginine-transporting tivity to these alkylating agents which is largely dependent on System. Biochim. Biophys. Acta, 127: 325-338, 1966. the guanine-cytosine level in the combination. The choice of tyro- 14. Haddow, A., and Ross, W. C. J. Tumour Growth Inhibitory sine and glycine for a comparative study was made with this in Alkyl Sulphonates. Nature, 177: 995, 1956. mind. The triplet requirement for tyrosine transfer is UA (U, 15. Jackson, H., Fox, B. W., and Craig, A. W. Antifertility Sub stances arid their Assessment in the Male Rodent. J. Reprod. C) and that for glycine is GG (U, C, A, G). Although only 2 Fertility, 2: 447-465, 1961. neutral amino acids have been considered in this paper, their 16. Jacquez, J. A. The Kinetics of Carrier-mediated Active Trans relative sensitivity to inhibition would support this suggestion. port of Amino Acids. Proc. Nati. Acad. Sci. U. S., 47: 153- The dose levels for 50% inhibition of the amino acid uptake into 163, 1961. protein for these 2 amino acids were 1.4 IMI for glycine and 2.2 17. Lawley, P. D., and Brookes, P. Further Studies on the Alkyla- mil for tyrosine. Clearly, other amino acids will require to be tion of Nucleic Acids and Their Constituent Nucleotides. investigated in a simpler system before this mechanism can be Biochem. J., 89: 127-138, 1963. considered to be operating. 18. Lawley, P. D., and Brookes, P. Molecular Mechanisms of the Cytotoxic Action of Difunctional Alkylating Agents and of Resistance to This Action. Nature, 206: 480-483, 1965. REFERENCES 19. Lowry, O. IL, Rosebrough, N. J., Farr, A. L., and Randall, 1. Bubula, B., and Holdsworth, E. S. Protein Synthesis in Bacil R. J. Protein Measurement with the Folin Phenol Reagent. lus stcarothermophilus. Biochim. Biophys. Acta, 1S3: 376-389, J. Biol. Chem., 193: 265-275, 1951. I960. 20. Magee, P., and Farber, E. Toxic Liver Injury and Carcino- 2. Burton, K. A. Study of the Conditions and Mechanism of the genesis. Methylation of Rat Liver Nucleic Acids by Dimethyl- Diphenyl Amine Reaction for the Colorimetrie Estimation of nitrosamine in vivo. Biochem. J., 83: 114-124, 1962. Deoxyribonucleic Acid. Biochem. J., 68: 315-323, 1956. 21. Mailer, R. K., and Heidelberger, C. Studies on OPSPA. IV. 3. Ceriotti, G. Determination of Nucleic Acids in Animal Tis Metabolism of OPSPA in the Rat and Human. Cancer Res., sues. J. Biol. Chem., 214: 59-70, 1955. 17: 296-301, 1957. 4. Christensen, H. N. Reactive Sites and Biological Transport. 22. Moore, L. D., and Umbreit, W. W. Membrane Associated Pro Advan. Protein Chem., IS: 239-314, 1960. tein Synthesis in Streptococcus faecalis. Biochim. Biophys. Acta, 5. Christensen, H. 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Brian W. Fox and Margaret Fox

Cancer Res 1967;27:2234-2239.

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